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Abstract:

The present invention provides a method to diagnostically detect the
variants of a given pathogen, such as HIV, hepatitis C, hepatitis B
(HBV), Parvovirus B19, etc., with the use of a single detection probe.

Claims:

1. A kit for the detection of a target nucleic acid molecule in a sample,
wherein the target nucleic acid molecule is known to have variant
sequences, comprising: (i) a set of nose-to-nose primers comprising a
forward primer and a reverse primer, each comprising a sequence of
nucleotides and wherein the forward and reverse primers are characterized
in that a nucleotide at the 3' end of the reverse primer hybridizes with:
(i) a nucleotide at the 5' end of a forward primer extension product or;
(ii) a nucleotide separated from the nucleotide at the 5' end of the
forward primer extension product by a gap; and a nucleotide at the 3' end
of the forward primer hybridizes with: (i) a nucleotide at the 5' end of
a reverse primer extension product or; (ii) a nucleotide separated from
the nucleotide at the 5' end of the reverse primer extension product by
the gap; (ii) reagents for performing a primer extension chain reaction
and; (iii) a self-altering signal-generating probe which detects the
presence of primer amplification products, wherein the probe comprises a
first nucleic acid sequence attached to a reporter moiety capable of
generating a detectable signal; a second nucleic acid sequence attached
to an interactive moiety capable of altering the signal of the reporter
moiety; and a probe sequence which connects the first and second nucleic
acid sequences.

2. The kit according to claim 1, wherein the target nucleic acid molecule
is a virus selected from the group consisting of human immunodeficiency
virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV).

3. The kit according to claim 1, wherein the gap comprises a highly
conserved region of the genome of the virus.

4. The kit according to claim 1, wherein the gap comprises from about one
to about five nucleotides.

5. The kit according to claim 1, wherein the nucleic acid molecule which
is complementary to the target nucleic acid molecule of step (d)(i) is
provided separately as the cDNA of the target nucleic acid molecule.

6. The kit according to claim 1, wherein the second nucleic acid sequence
of the self-altering signal-generating probe is hybridized to the first
nucleic acid sequence of the self-altering signal-generating probe and
wherein the probe sequence comprises either: (a) the nucleotide sequence
of a segment of the forward primer; or (b) the nucleotide sequence of a
segment of the reverse primer; and wherein upon contacting the
amplification products with the probe, the probe sequence of the probe
hybridizes with the additional reverse primer amplification product or
with the additional forward primer amplification product, and the first
and second nucleic acid sequences become denatured, thereby generating a
signal by the reporter moiety; and wherein the signal generated by the
reporter moiety indicates the presence of the target molecule.

7. The kit according to claim 6, wherein the probe sequence comprises
either: (a) the nucleotide sequence of a segment of the forward primer,
and not the nucleotide sequence of a segment which is complementary to
the reverse primer; or (b) the nucleotide sequence of a segment of the
reverse primer, and not the nucleotide sequence of a segment which is
complementary to the forward primer.

8. The kit according to claim 6, wherein the probe sequence comprises
either: (a) the nucleotide sequence of a segment of the forward primer
and the nucleotide sequence of a segment which is complementary to the
reverse primer; or (b) the nucleotide sequence of a segment of the
reverse primer and the nucleotide sequence of a segment which is
complementary to the forward primer.

9. The kit according to claim 6, wherein the level of the detectable
signal generated by the probe is proportional to the quantity of the
target nucleic acid molecule in the sample.

10. The kit according to claim 8, wherein about sixty to about
ninety-five percent of the probe sequence comprises either: (i) the
nucleotide sequence of a segment of the forward primer; or (ii) the
nucleotide sequence of a segment of the reverse primer.

11. The kit according to claim 6, wherein: if the probe sequence
comprises the nucleotide sequence of a segment of the reverse primer, the
molar ratio of the reverse primer to the forward primer is in the range
from about 1:5 to about 1:20; or if the probe sequence comprises the
nucleotide sequence of a segment of the forward primer, the molar ratio
of the forward primer to the reverse primer is in the range from about
1:5 to about 1:20.

12. The kit according to claim 6, wherein the probe sequence comprises
from about ten to about thirty nucleotide residues.

13. The kit according to claim 9, wherein the probe sequence comprises
from about eighteen to about twenty-four nucleotide residues.

14. The kit according to claim 6, wherein the detectable signal is a
luminescent signal.

15. The kit according to claim 14, wherein the luminescent signal is a
fluorescent signal or a chemiluminescent signal.

16. The kit according to claim 6, wherein the reporter moiety is attached
at the 5' terminus or 3' terminus of the self-altering signal-generating
probe.

17. The kit according to claim 6, wherein the interactive moiety is
attached at the 5' terminus or 3' terminus of the self-altering
signal-generating probe.

18. The kit according to claim 6, wherein the reporter moiety is a
fluorophore selected from the group consisting of a xanthene dye, a
cyanine dye, a dansyl derivative, EDANS, coumarin, Lucifer yellow,
BODIPY, Cy3, Cy5, Cy7, Texas red, erythrosine, naphthylamine, Oregon
green, or combinations thereof.

19. The kit according to claim 6, wherein the interactive moiety is a
quencher or a fluorophore.

20. The kit according to claim 19, wherein the quencher is DABCYL,
anthroquinone, nitrothiazole, nitroimidazole or malachite green.

21. The kit according to claim 1, wherein the amplification products are
measured and quantitated by end-point analysis or by real-time analysis.

22. The kit according to claim 1, wherein the amplification products are
measured using a standard curve derived from a series of threshold cycle
measurements.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser.
No. 12/011,603 filed Jan. 28, 2008, which is a division of U.S. patent
application Ser. No. 10/399,843, filed Sep. 2, 2003, now U.S. Pat. No.
7,348,164, which is an application under 371 of International Patent
Application PCT/US2002/12035 filed Apr. 17, 2002, which in turn claims
the benefit of U.S. Provisional Application No. 60/284,334, filed Apr.
17, 2001, which is incorporated herein by reference. The entire contents
of each of these applications is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The detection of closely related genetic variants is a significant
challenge of analytical diagnostics. Pathogens such as, for example,
viruses and bacteria, generally mutate frequently and form such genetic
variants.

[0003] For example, the nucleic acid sequences of human immunodeficiency
virus (HIV-1) having different origins, are different from each other.
The different types of HIV-1 are divided into groups and subtypes. The
major group M consists of ten currently identified subtypes, designated
as subtypes A through H, J and K. In addition to M-group viruses, two
other groups, N and O, have been identified (Simon et al, 1998, Nature
Med, 4:1032-1037). Within groups and subtypes, new strains of the virus
are continuously being generated due to the error-prone nature of the
HIV-1 replicative machinery.

[0004] Similarly, hepatitis C virus (HCV) does not exist as a homogeneous
RNA population. Even within a single infected individual, numerous
heterogeneous viral genomes (quasispecies) may co-exist. In addition,
multiple genotypes of HCV have been identified on the basis of nucleotide
sequence analysis of viral variants isolated from different geographic
regions. There are currently six main HCV genotypes, classified
numerically from 1 to 6. Genotypes are further subdivided according to
subtype.

[0005] Due to this genetic variation of pathogens within a species, the
range of diagnostic tests that provide reliable results are highly
limited. Most detection methods currently available for detecting
pathogens in a sample are based either on the detection of the pathogens'
antigens, pathogen-induced antibodies, or the pathogens' intrinsic
enzymes, e.g. intrinsic HIV reverse transcriptase. In addition to being
inconvenient, such methods are frequently not very sensitive. For
example, the method currently implemented by blood banks for screening of
HIV-1 infection in blood donors is the detection of antibodies to virus
proteins. This method fails to detect individuals in the early acute
phase of the infection who have not yet developed diagnostic antibodies
to the virus.

[0006] Screening methods that are based on the detection of nucleic acid
sequences are sensitive and convenient. However, these tests may not
always be reliable for detection of closely related genetic variants.

[0007] One of the currently available nucleic acid sequence-based
detection methods utilizes molecular beacons (Tyagi and Kramer, 1996,
Nat. Biotechnol., 14(3):303-308). Molecular beacons are single-stranded
oligonucleotide probes that have a stem-loop structure. (See FIG. 1.) The
loop portion of the molecule is a probe sequence complementary to a
target nucleic acid molecule. The stem is formed by the annealing of
complementary arm sequences on the ends of the probe sequence. A
fluorescent moiety is attached to the end of one arm; and a quenching
moiety is attached to the end of the other arm. The hybridization of the
arms of the stem to each other keeps these two moieties in close
proximity, causing the fluorescence of the fluorophore to be quenched by
energy transfer (FIG. 1a). In the presence of the beacon's complementary
DNA target, the loop structure hybridizes to the target, preventing the
arms of the stem from remaining hybridized. The fluorophore and quencher
are physically separated, and fluorescence is obtained (FIG. 1b).

[0008] Molecular beacons are currently used for real-time quantitative
PCR. PCR primers are designed to amplify a specific segment of DNA,
usually less than 200 base pairs in length. The beacon is typically
designed so that its loop is complementary to a short (20-25 b.p.) region
on one of the amplified DNA strands. The complementary region of these
amplified DNA strands is the portion of these strands which has been
added to the primers.

[0010] To date, this sensitivity to sequence variation has severely
limited the application of molecular beacon technology to the diagnosis
of viral infection. The molecular beacons cannot efficiently detect the
variant sequences of DNA or RNA targets. For example, a beacon designed
to recognize PCR product from HIV strain A may not recognize PCR product
from HIV strain B. (See FIG. 2.)

[0011] Thus, the present technology would require several different
beacons to allow for the detection of all the different genotypes of the
virus. That is, even though some highly conserved regions of the genome
of HIV-1 are known to exist, it is likely that several different beacons
would be needed to detect all the known subtypes of this virus. Moreover,
even with the use of several different beacons, other variants of HIV-1
that have not been identified may not be detected.

[0012] Thus, current technology does not provide a convenient or efficient
diagnostic assay for detection of all related genetic variants of
pathogens.

[0013] There is an urgent need for sensitive, convenient nucleic
acid-based screening assays capable of detecting closely related genetic
variants. For example, there is a need for assays capable of detecting
viruses, bacteria and other pathogens, directly in contaminated blood.
Such assays are needed to detect blood or plasma units from individuals
in the early acute stages of a pathogen infection, i.e., before the
individual has developed diagnostic antibodies to the virus.

[0014] Accordingly, one of the purposes of the present invention is to
overcome the above limitations in the prior art by providing a convenient
and efficient diagnostic assay for detection of multiple variants of a
particular target nucleic acid molecule.

SUMMARY OF THE INVENTION

[0015] These and other objects, as will be apparent to those having
ordinary skill in the art, have been met by providing a method to
diagnostically detect the variants of a given pathogen, such as HIV,
hepatitis C, hepatitis B (HBV), Parvovirus B19, etc., with the use of a
single detection probe, i.e., a universal multi-variant detection system.
In one embodiment, the single detection probe is a molecular beacon.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1: A graphical illustration of a molecular beacon. At the
appropriate annealing temperature the beacon will either: (A) in the
absence of a complementary target sequence, form a stem loop structure
causing the quenching moiety (quadrature) to quench the luminescence of
the reporter moiety (O); or (B) in the presence of a complementary
target, bind to the target allowing the reporter to emit its signal.

[0017] FIG. 2: Conventional PCR using molecular beacons. PCR primers are
designed to amplify a segment of viral RNA. A molecular beacon is
designed so that its probe loop will hybridize to a segment of the PCR
product which is internal to the two PCR primers. The beacon is capable
of hybridizing to PCR product of virus strain A, but fails to detect PCR
product of virus strain B because of mismatches in the target sequence
(shown in lower case).

[0018] FIG. 3: A graphical illustration of one of the principles of the
invention. (a.) Reverse and forward PCR primers (>30 b.p.) are
designed to hybridize directly "nose-to-nose" to the target RNA (or DNA)
and its complementary DNA strand respectively, such that the generated
PCR product possesses no intervening sequence. The target-specific loop
of the molecular beacon is designed to hybridize to the DNA sequence
created by the junction of one of the primers and the other primer's
complement. PCR primers will hybridize to target templates with
mismatched residues, indicated by "X." Dotted lines indicate
hybridization. (b.) The DNA sequence of the PCR product amplified from
all templates is identical to the combined sequence of one of the primers
and the other primer's complement. The molecular beacon is thus capable
of hybridizing to PCR products generated from all templates.

[0019] FIG. 4: An example of the method. An amplification of different
subtypes of HIV using "nose-to-nose" primers and a molecular beacon
designed to recognize a sequence created by the junction of one of the
primers and the other primer's complement. Mismatches between the
sequence of the HIV variants and either the primers or beacon loop are
shown in lower case boldface.

[0020] FIG. 5: Illustration of Variations on Primer Location. (A). The
beacon loop can be designed to hybridize to an amplified sequence created
equally by the two PCR primers as shown in (I). Alternatively, the beacon
can be designed to hybridize "asymmetrically" to an amplified sequence
created primarily by either the forward or reverse primer as shown in
(II) to (IV). (B). In a further variation, the forward and reverse
primers are separated by a nucleotide gap which corresponds to a highly
conserved region of the viral genome.

[0021] FIG. 6: DNA sequence alignment of the V3 loop and flanking regions
of four variants of HIV showing the positions of molecular beacon and
primers for both conventional and "nose-to-nose" PCR (a). The protein
coding strand of HIV/RT-1 is aligned with that of 3 other virus variants,
HIV/RT-10, HIV-38-1 and HIV/38-3. Mismatches to the sequence of HIV/RT-1
and to the molecular beacon are shown in lower case in bold. The relative
location of forward and reverse primers for conventional PCR are
indicated by dotted (. . . . . .) and dashed (- - - -) lines
respectively. The relative location of forward and reverse primers for
nose-to-nose PCR are indicated by double (=) and solid (--) lines
respectively. The location of the beacon probe is shown above the
sequence. All primers and the beacon probe sequences are derived from the
sequence of HIV/RT-1. (b). Structure of the molecular beacon. The probe
loop is shown in upper case, the complementary stem nucleotides are shown
in lower case. The fluorophore FAM is conjugated to the 5' end, the
quencher DABCYL is conjugated to the 3' end.

[0024] FIG. 9: An example of a standard curve for quantitation of HCV RNA
using "nose-to-nose" RT-PCR with product detection using a molecular
beacon. (a). "Nose-to-Nose" RT-PCR for HCV RNA was performed with an
input of 0, 10, 25, 50, 100, 103, 104, 105 or 106
synthetic HCV RNA molecules per RT-PCR reaction. Change in fluorescence
(delta Rn) was measured at the annealing temperature for each PCR cycle
in the ABI 7700 Sequence Detector. Threshold values (Ct) were then
calculated using software provided with the instrument. (b). The RNA copy
number of each standard sample is plotted against its Ct value ( ). The
Ct values for unknown test samples (∘) are plotted against
the standard curve and RNA copy number is extrapolated from the X-axis.

[0025] FIG. 10: A step-by-step illustration of an amplification reaction
of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides a method for determining the
presence of a target nucleic acid molecule in a biological sample with
the use of a single detection probe.

[0027] The method comprises the amplification of a target nucleic acid
molecule by means of a primer extension chain reaction wherein the
primers of the reaction are a set of "nose-to-nose" primers, including a
forward and reverse primer, as described below. (See FIGS. 3 and 4.)

[0028] The target nucleic acid molecule is a nucleic acid molecule whose
full or partial sequence is sufficiently known to make primer extension
chain reaction primers. The target nucleic acid molecule can be single or
double stranded.

[0029] The target nucleic acid molecule exists as a family of highly
homologous sequences. These different sequences within a family are
referred to as variants. The origin of the variants include, for example,
gene mutations and polymorphisms.

[0030] Nucleic acid molecules which are known to have variants include,
for example, viruses and bacteria. Examples of viruses include HIV, HCV,
HBV and human parvovirus B19. Examples of bacteria include E. coli, S.
pneumoniae, N. meningitidis, N. gonorrhoeae, M. tuberculosis and Borrelia
species (Lyme disease).

[0031] A biological sample from which a target nucleic acid molecule can
be detected is any bodily fluid, cells or cellular debris. Examples of
biological samples include blood, serum, semen, mucous or other bodily
exudates.

[0032] The present invention can be used in any type of primer extension
chain reaction that leads to the amplification of the target nucleic acid
molecule or a subregion of this molecule. Amplification reactions
include, for example, the polymerase chain reaction (PCR), including
quantitative PCR; strand displacement amplification (SDA); transcription
mediated amplification (TMA); and nucleic acid sequence based
amplification (NASBA). NASBA amplifies RNA. NASBA is described in EP-A-0
329 822.

[0033] The conventional polymerase chain reaction (PCR) amplification
process is well known in the art. Conditions suitable for carrying out a
polymerase chain reaction are described in U.S. Pat. Nos. 4,683,195;
4,683,202; and 4,965,188. Commercial vendors, such as Perkin Elmer
(Norwalk, Conn.), market PCR reagents and publish PCR protocols. A PCR
amplification reaction mixture contains reagents necessary to carry out
an amplification reaction. Typically, the mixture contains an agent for
polymerization, such as thermostable DNA polymerase; deoxynucleoside 5'
triphosphates (dNTP's); and a divalent metal cation in a suitable buffer.

[0034] Either DNA or RNA target sequences can be amplified by the methods
of the present invention. In the case of PCR amplification of an RNA
target, such as a viral genomic nucleic acid, the first step is the
synthesis of a DNA copy (cDNA) of the target sequence. The reverse
transcription can be carried out as a separate step or, preferably, in a
combined reverse transcription-polymerase chain reaction (RT-PCR). The
RT-PCR amplification of RNA is well known in the art and described in
U.S. Pat. Nos. 5,322,770 and 5,310,652; Myers and Gelfand, 1991,
Biochemistry 30(31):7661-7666; U.S. Pat. No. 5,527,669; Young et al.,
1993, J. Clin. Microbiol. 31(4):882-886; and Young et al., 1995, J. Clin.
Microbiol. 33(3):654-657.

[0035] Primers are also included in the PCR reaction mixture. A primer is
an oligonucleotide which, upon hybridizing to a template nucleic acid
molecule, is capable of acting as a point of synthesis initiation during
an amplification reaction. The template nucleic acid is the initial
target nucleic acid molecules; and the amplification products generated
from these molecules.

[0036] The length of the primers of the present invention is not critical.
Typically the primer length ranges from about 15 to 55 nucleotides; more
typically from about 20 to 45 nucleotides; and most typically from about
25 to 35 nucleotides. Preferably, the primers are constructed to be
relatively long (>30 bases) to maximize the number of mismatches that
can be tolerated between a primer and its template. A primer pair need
not be of the same length. For example, the forward primer may be made up
of twenty-nine nucleotides; while the reverse primer can be made up of
twenty-two nucleotides.

[0037] The primers can be natural or synthetic. For PCR, the primers are
preferably single-stranded oligodeoxyribonucleotides.

[0038] Hybridization refers to the formation of a duplex structure by two
single-stranded nucleic acids due to complementary base pairing.
Hybridization can occur between fully complementary nucleic acid strands
or between "substantially complementary" nucleic acid strands that
contain minor regions of mismatch, i.e. variants. The degree of mismatch
tolerated can be controlled by suitable adjustment of the hybridization
conditions. Conditions under which only fully complementary nucleic acid
strands will hybridize are referred to as "stringent hybridization
conditions" or "sequence-specific hybridization conditions." Stable
duplexes of substantially complementary sequences can be achieved under
less stringent hybridization conditions.

[0039] The hybridization conditions, i.e. stringency, of the present
invention are set so that the primers can tolerate mismatches between the
primers and the template, thereby allowing hybridization to all genetic
variants. For example, the conditions could be set so that hybridization
between a primer and a template can occur with up to 20% of the base
pairs between the primer and the template mismatched.

[0041] A detailed description of a cycle of a primer extension chain
reaction of the present invention follows. The specific reaction
described is PCR. However, other types of primer extension chain
reactions can be used in the methods of this invention.

[0042] FIG. 10 gives a step-by-step illustration of an amplification
reaction of the invention. The target nucleic acid molecule is
represented by T. The X's within the target sequence represent sites of
potential variations.

[0043] The target nucleic acid molecule can exist as a single-stranded
molecule, or as part of a double stranded molecule. In the example
illustrated in FIG. 10, the target nucleic acid molecule is double
stranded. TC represents a nucleic acid molecule which is complementary to
T.

[0044] As in conventional PCR, in each cycle of the amplification reaction
any double-stranded nucleic acid molecules in a sample are rendered
single-stranded by denaturation. Hybridization then takes place between
the primers and the target nucleic acid molecules. FIG. 10(a) illustrates
hybridization between a reverse primer (RP) and a target nucleic acid
sequence (T).

[0045] As shown in FIG. 10(b), the reverse primers then are extended,
using the target nucleic acid molecules as templates, to form reverse
primer amplification products (RPA). The reverse primer amplification
products (RPA) are comprised of the reverse primer (RP) joined to the
reverse primer extension product (RPE). For the purposes of this
specification, the reverse primer extension product is the nucleic acid
segment which is added to the reverse primer.

[0046] As can be seen from FIG. 10(b), some of the variations (X)
contained in the target sequence do not appear in the RPA. Specifically,
the portion of the RPA which is made up of the RP does not contain
variations.

[0047] As shown in FIG. 10(c), the reverse primer amplification products
formed in step (b) are denatured from their templates.

[0048] The forward primers (FP) are hybridized to either: (i) nucleic acid
molecules which are complementary to the target nucleic acid molecules
(TC), if present; or (ii) the reverse primer amplification products
(RPA). FIG. 10(d) illustrates the former embodiment. Nucleic acid
molecules complementary to the target nucleic acid molecules, would be
present if the target nucleic acid molecules were part of a
double-stranded molecule.

[0049] The forward primers are then extended, using as templates the
complementary nucleic acid molecules (TC) or using the reverse primer
amplification products (RPA). FIG. 10(e) illustrates the former
embodiment.

[0050] As shown in FIG. 10(e), the forward primers are extended to form
forward primer amplification products (FPA). The forward primer
amplification product (FPA) is comprised of the forward primer (FP)
joined to the forward primer extension product (FPE). For the purposes of
this specification, the forward primer extension product is the nucleic
acid segment which is added to the forward primer.

[0051] As shown in FIG. 10(e), when compared with the target sequence,
some of the variations (X) contained in the target sequence do not appear
in the FPA. Specifically, the portion of the FPA which is made up of the
FP does not contain variations.

[0052] As shown in FIG. 10(f), the forward primer amplification products
formed in FIG. 10(e) are denatured from their templates.

[0053] As shown in FIG. 10(g), the reverse primers hybridize to the FPE
portion of the FPA.

[0054] As shown in FIG. 10(h), the reverse primers are extended, using the
FP portion of the FPA as templates, to form additional reverse primer
amplification products (ARPA), wherein a reverse primer joined to an
additional reverse primer extension product (ARPE) constitutes an ARPA.

[0055] As shown in FIG. 10(i), the ARPA products are denatured from their
templates.

[0056] As shown in FIG. 10(j), the forward primers hybridize to the RPE
portion of the RPA.

[0057] As shown in FIG. 10(k), the forward primers are extended, using the
RP portion of the RPAs as templates, to form additional forward primer
amplification products, wherein a forward primer joined to an additional
forward primer extension product constitutes an AFPA.

[0058] As shown in FIG. 10(l), the AFPA products are denatured from their
templates.

[0059] Steps (g) to (l) are repeated, using the additional reverse primer
amplification products and the additional forward primer amplification
products as templates for the reverse and forward primers, a sufficient
number of times to produce a detectable quantity of additional reverse
primer amplification product and/or of additional forward primer
amplification product. Preferably, the steps are repeated using an
automated cycling instrument. A sufficient number of times is at least
about ten times, preferably at least about twenty times; more preferably
at least about thirty times; and most preferably at least about forty
times.

[0060] The present inventors have discovered advantages when the primers
hybridize with the amplification products in a certain way, which the
inventors refer to as "nose-to-nose."

[0061] As can be seen in FIG. 10(g), the sequence of the forward primer
amplification products and the additional forward primer amplification
products are such that the nucleotide at the 3' end of the reverse primer
hybridizes with the nucleotide at the 5' end of the forward primer
extension product or of the additional forward primer extension product.

[0062] Analogously, as can be seen in FIG. 10(j), the sequence of the
reverse primer amplification products and the additional reverse primer
amplification products are such that the nucleotide at the 3' end of the
forward primer hybridizes with the nucleotide at the 5' end of the
reverse primer extension product or of the additional reverse primer
extension product.

[0063] FIG. 10 illustrates the primer extension chain reaction of only one
variant. As indicated above, all variants of a family of pathogens can be
amplified by the method of the invention.

[0064] The additional amplification products are identical, regardless of
which variant they were generated from. Thus, in the example shown in
FIG. 10, the additional reverse primer amplification products have the
sequence of the reverse primer directly joined to the additional reverse
primer extension product. The additional reverse primer extension product
is complementary to the forward primer (the forward primer complement).
See FIG. 10(h).

[0065] Analogously, the additional forward primer amplification products
have the sequence of the forward primer directly joined to the additional
forward primer extension product. The additional forward primer extension
product is complementary to the reverse primer (reverse primer
complement). See FIG. 10(k).

[0066] Therefore, all of the additional primer amplification products have
sequences that are combinations of either the reverse primer and forward
primer complement, or the reverse primer complement and the forward
primer. Since the reverse and forward primers all have the same
sequences, all of the additional primer amplification products have the
same sequences. In other words, all of the potential variations have been
eliminated.

[0067] In another embodiment, the sequence of the forward primer
amplification products and the additional forward primer amplification
products are such that the nucleotide at the 3' end of the reverse primer
hybridizes with a nucleotide separated from the nucleotide at the 5' end
of the forward primer extension product or of the additional forward
primer extension product by a gap of nucleotides. Analogously, the
sequence of the reverse primer amplification products and the additional
reverse primer amplification products are such that the nucleotide at the
3' end of the forward primer hybridizes with a nucleotide separated from
the nucleotide at the 5' end of the reverse primer extension product or
of the additional reverse primer extension product by a gap of
nucleotides.

[0068] In both cases, the gap comprises a sequence known to be highly
conserved. Highly conserved regions of the genomes of viruses and
bacteria are known. For example, in the published sequence of HCV, short
stretches of nucleic acids in the 5' non-coding region of the viral
genome are known to be highly conserved between HCV genotypes (Okamoto et
al., J. Gen. Virol., 1991, 2697-2704; Smith et al., J. Gen. Virol., 1995,
76:1749-1761; Simmonds et al., J. Gen. Virol., 1993, 74: 2391-2399).

[0069] The gap preferably contains no more than five nucleotides. If the
gap contains two to five nucleotides, one or two of the nucleotides can
be mismatched and still hybridize with the probe sequence. If the gap
contains one nucleotide, this nucleotide can be a mismatch. Preferably,
there are no mismatches.

[0070] Once the amplification reaction has been completed, the presence of
the additional reverse primer amplification products or the additional
forward primer amplification products is detected by methods known in the
art.

[0071] Preferably, the method of detection is based on the detection of
the nucleic acid sequences of the additional reverse primer amplification
products or of the additional forward primer amplification products. The
detection probe used in such a method comprises a sequence that is
capable of hybridizing with the additional reverse primer amplification
products or with the additional forward primer amplification products.
Since these amplification products are identical, only one detection
probe is needed to reliably detect all the amplification products.

[0072] Additionally, the identity of the amplification products allows for
stringent hybridization conditions to be used during the hybridization
required for detection. The use of stringent conditions leads to more
reliable results by reducing the possibility of false positives from
coincidentally similar non-target sequences.

[0073] The detection probe can be DNA, RNA, or combinations thereof.
Modified nucleotides may be included, for example peptide nucleic acid
(PNA), nitropyrole-based nucleotides, or 2'-O-methylribonucleotides. The
nucleosides of the nucleic acid or modified nucleic acid molecules may be
linked in the usual manner, i.e. through phosphate linkages.
Alternatively, the nucleosides may be linked through modified linkages,
for example phosphorothioates.

[0074] In one embodiment, the probe sequence hybridizes over the junction
in the amplification products. That is, the probe sequence hybridizes to
a portion of both the FP sequence and the AFPE sequence of the AFPA; or
the probe sequence hybridizes to a portion of both the RP sequence and
the ARPE sequence of the ARPA.

[0075] In this embodiment, the probe sequence is comprised of the
nucleotide sequence of a segment of one of the primers and the nucleotide
sequence of a segment which is complementary to the other primer. More
specifically, the probe sequence comprises either: (i) the nucleotide
sequence of a segment of the forward primer and the nucleotide sequence
of a segment which is complementary to the reverse primer; or (ii) the
nucleotide sequence of a segment of the reverse primer and the nucleotide
sequence of a segment which is complementary to the forward primer. The
probe sequence can comprise either whole or partial sequences of the
primer, and the other primer's complement.

[0076] The probe sequence can be made up of equal portions of the
nucleotide sequence of one of the primers and the nucleotide sequence of
the other primer's complement. In such a case, the probe sequence will
"hybridize symmetrically" to the amplification products. (See FIG.
5A(I).)

[0077] Preferably, for improved sensitivity, the probe sequence can be
designed to "hybridize asymmetrically" to the amplification products. In
particular, the probe sequence can be made up of unequal portions of the
nucleotide sequence of one of the primers and the nucleotide sequence of
the other primer's complement. (See FIG. 5A(II-IV).) For example, about
60% to about 99% of the probe sequence comprises the nucleotide sequence
of one of the primers. The remainder of the probe sequence (e.g., about
1% to about 40%) comprises the nucleotide sequence of the complement of
the other primer. More preferably, the percentage of the probe sequence
which corresponds to the sequence of one of the primers is from about 80%
to about 97%.

[0078] Where the sequence of the additional amplification products
contains a segment of one of the primers directly adjoined to a segment
of the other primer's complement, the probe sequence can be designed to
hybridize exactly to all, or a portion of, the additional amplification
products.

[0079] Where the additional amplification products contain an intervening
gap, the gap is preferably a known sequence so that the probe sequence
can be designed to be fully complementary to the gap. However, a probe
sequence can hybridize to additional amplification products which contain
a certain number of mismatched residues in the gap, as described above.

[0080] Instead of hybridizing over the junction, the probe sequence can
also hybridize exclusively with the segment of the additional
amplification product that is complementary to a primer. Accordingly, in
this embodiment, the probe sequence is comprised of either: the
nucleotide sequence of a segment of the forward primer, and not the
nucleotide sequence of a segment which is complementary to the reverse
primer; or the nucleotide sequence of a segment of the reverse primer,
and not the nucleotide sequence of a segment which is complementary to
the forward primer.

[0081] In one embodiment of the primer extension chain reactions of the
invention, equal concentrations of the forward primer and reverse primer
are used. In this embodiment, the concentrations are said to be
symmetric.

[0082] In a preferred embodiment, "asymmetric concentrations" of the
forward and reverse primers are used. In particular, for improved
sensitivity, the primer whose nucleotide sequence makes up part of the
probe sequence is provided in a lower concentration in the sample as
compared with the other primer. "Asymmetric concentrations" of primers
are particularly preferred if the probe sequences "hybridize
asymmetrically" with the additional amplification products; or hybridize
exclusively with the segment of the additional amplification products
that is complementary to a primer.

[0083] For example, if the probe sequence comprises a segment of the
nucleotide sequence of the forward primer, then the molar ratio of the
forward primer to the reverse primer (FP:RP) is about 1:5 to about 1:20.
Analogously, if the probe sequence comprises a segment of the nucleotide
sequence of the reverse primer, then the molar ratio of the reverse
primer to the forward primer (RP:FP) is about 1:5 to about 1:20, more
preferably from about 1:6 to about 1:15, and most preferably about 1:10.

[0084] After the amplification reaction has taken place in the biological
sample a sufficient number of times, the detection probe is contacted
with the sample. The probe sequence of the detection probe hybridizes
with any additional amplification products that may be present in the
sample. If the probe sequence comprises the nucleotide sequence of a
segment of the forward primer (exclusively or further comprising the
nucleotide sequence of a segment of the reverse primer complement), the
probe sequence will hybridize with the additional reverse primer
amplification products. Analogously, if the probe sequence comprises the
nucleotide sequence of a segment of the reverse primer (exclusively or
further comprising and the nucleotide sequence of a segment of the
forward primer complement), the probe sequence will hybridize with the
additional forward primer amplification products.

[0085] In a preferred embodiment, the detection probe is a self-altering
signal-generating probe. This probe comprises a first nucleic acid
sequence; a second nucleic acid sequence complementary to the first
nucleic acid sequence; and a probe sequence which connects the first
nucleic acid sequence with the second nucleic acid sequence. The first
nucleic acid sequence is attached to a reporter moiety which is capable
of generating a detectable signal. The second nucleic acid sequence is
attached to an interactive moiety which is capable of altering the signal
generated by the reporter moiety when the reporter moiety and the
interactive moiety are in sufficient proximity to each other. For
example, when the first and the second nucleic acid sequences are
hybridized to one another, known as the "closed conformation," the
reporter moiety is brought into proximity with the interactive moiety.
Therefore, the signal is altered. Altering the signal includes
decreasing, i.e. quenching; increasing; or otherwise changing the signal,
such as the intensity or wavelength of the signal. Quenching the signal
includes reducing or eliminating the signal.

[0086] The reporter and interactive moieties can be attached at any point
on the detection probe which would allow for the alteration by the
interactive moiety of a signal generated by the reporter moiety for
detection of the additional amplification products. In the preferred
embodiment, the reporter moiety and the interactive moiety are attached
at the distal termini of the self-altering signal-generating probe.

[0087] In the absence of additional amplification products, the detection
probe is in the closed conformation. The reporter and interactive
moieties are in proximity to each other. Therefore, the signal is
altered.

[0088] Upon hybridization of the probe sequence with the additional
reverse primer amplification product or with the additional forward
primer amplification product, the first and second nucleic acid sequences
of the detection probe become denatured. This is known as the "open
conformation."

[0089] Upon denaturation, the interactive moiety is no longer in
sufficient proximity to the reporter moiety to alter the signal. The
difference between the altered signal and the unaltered signal is
detected. When the interactive moiety quenches the signal, for example,
the unaltered signal is increased; or if the quenching was complete, a
signal is generated.

[0090] The strength of the hybridization formed between the first and
second nucleic acids (i.e., the stem of the probe) can be adjusted by
routine experimentation to achieve proper functioning. For example, the
strength is a function of the length of the nucleotides. The lengths of
the first and second nucleic acid sequences are preferably in the range
of about 3 to 15, more preferably about 4 to 7 nucleotides. In addition
to length, the strength of the hybridization can be reduced by decreasing
the G-C content and by inserting destabilizing mismatches in the
nucleotides.

[0091] The length of the probe sequence is not critical. However, the
length cannot be so short that effective binding with the additional
amplification products is not achieved. Additionally, the length cannot
be so great that separation of the reporter moiety and the interactive
moiety is not achieved despite the probe sequence being hybridized with
the products. Preferably, the probe sequence comprises from about ten to
about thirty nucleotides; more preferably from about eighteen to about
twenty-four nucleotides; and most preferably from about nineteen to about
twenty-two nucleotides. The probes can be free in solution, or they can
be tethered to a solid surface.

[0092] Any concentration of the detection probe that produces a detectable
signal can be used in the methods. For example, the concentration of the
detection probe can be provided in the sample at about the same
concentration as one, or both, of the primers.

[0093] Preferably, the concentration of the detection probe is greater
than the concentrations of the primers. In this manner, the detection
probe is favored in the competition between the primer, whose nucleic
acid sequence is part of the probe sequence, and the detection probe for
the additional amplification products. This increase in the concentration
of the detection probe is particularly preferred when the probe sequence
"hybridizes asymmetrically" to the amplification products, or hybridizes
exclusively to the portion of the additional amplification products which
are complementary to a primer, as described above. For example, the
detection probe can be provided at a concentration which is from about
1.3 to about 5 times; more preferably from about 1.5 to about 3 times;
and most preferably about twice as great as the concentration of the
primer whose nucleic acid sequence is not part of the probe sequence.

[0094] In a preferred embodiment, the probe sequence is made up of, for
example, at least 65% of the sequence of the forward primer; the forward
primer is provided in a concentration which is about ten times less than
the concentration of the reverse primer; and the detection probe is
provided in a concentration which is about twice as great as the reverse
primer.

[0095] An unaltered signal generated by the reporter moiety is an
indication that the target nucleic acid molecule is present in the
sample. The level of the detectable unaltered signal generated by the
probe is proportional to the quantity of the target nucleic acid
molecules in the sample.

[0096] The detectable signal of the detection probes can be any kind of
signal including, for example, a luminescent signal, a color dye signal,
or a radioactive signal. In the preferred embodiment, the detectable
signal is a luminescent signal. The luminescent signal can be a
fluorescent signal or chemiluminescent signal.

[0097] In one embodiment, the reporter and interactive moieties of this
invention constitute a "FRET" pair. (Selvin, P. R., "Fluorescence
Resonance Energy Transfer," Methods in Enzymology 246: 300-335 (1995).)
FRET pairs rely on energy transfer for signal generation. The reporter
moiety absorbs energy at a first wavelength and emits a second, longer
wavelength. The interactive moiety absorbs some or most of the emitted
energy to the degree the interactive moiety's spectrum overlaps the
emission spectrum. If the interactive moiety is a quencher, the quencher
releases the energy as heat. If the interactive moiety is a fluorophore,
the interactive moiety re-emits at a third, still longer wavelength. The
mechanism of FRET-pair interaction requires that the absorption spectrum
of the interactive moiety overlaps the emission spectrum of the reporter
moiety. The efficiency of FRET interaction is linearly proportional to
that overlap

[0098] In another embodiment, the reporter moiety and interactive moiety
are a non-FRET pair. In particular, the interactive moiety need not have
an absorption spectrum that overlaps the emission spectrum of the
reporter moiety. That is, the absorption wavelength of the interactive
moiety can be shorter than the reporter's excitation maximum and emission
wavelength. Non-FRET pairs are described in U.S. Pat. No. 6,150,097 and
are incorporated herein by reference. The detectable signal in a non-FRET
pair can be a change in the absorption spectra, as an alternative to a
change in luminescence.

[0099] Preferably, the reporter moieties of the detection probes used in
the methods of this invention are fluorophores. The fluorophore can be a
xanthene dye, a cyanine dye, a dansyl derivative, EDANS, coumarin, such
as 3-phenyl-7-isocyanatocoumarin, Lucifer yellow, BODIPY, Cy3, Cy5, Cy7,
Texas red, erythrosine, naphthylamine, Oregon green, ALEXA fluor dyes,
acridines, such as 9-isothiocyanatoacridine and acridine orange,
N-(p-(2-benzoxazolyl)phenyl)maleimide, benzoxadiazoles, stilbenes, and
pyrenes.

[0100] The xanthene dye can be fluorescein or rhodamine. Preferably, the
fluorescein is 5-carboxyfluorescein (5-FAM); 6-carboxyfluorescein
(6-FAM); 2',4',1,4,-tetrachlorofluorescein (TET);
2',4',5',7',1,4-hexachlorofluorescein (HEX); eosin; calcium green;
fluorescein isothiocyanate (FITC); or NED. Preferably, the rhodamine dye
is tetramethyl-6-carboxyrhodamine (TAMRA);
tetrapropano-6-carboxyrhodamine (ROX);
2',7'dimethoxy-4',5'-dichloro-6-carboxyrhodamine (JOE) or
tetramethylrhodamine (TMR). Many suitable forms of these compounds are
commercially available with various substituents on their xanthene rings
which can be used as the site for bonding or as the bonding functionality
for attachment to an oligonucleotide.

[0101] The fluorophore can also be a naphthylamine compound. The
naphthylamine compounds have an amino group in the alpha or beta
position. Included among such naphthylamino compounds are
1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate
and 2-p-toluidinyl-6-naphthalene sulfonate.

[0102] The fluorophore can also be a combination fluorophore. An example
of a combination fluorophore are fluorescein-rhodamine dimers, described,
for example, by Lee et al. (1997), Nucleic Acids Research 25:2816.
Fluorophores may be chosen to absorb and emit in the visible spectrum or
outside the visible spectrum, such as in the ultraviolet or infrared
ranges.

[0103] Preferably, the interactive moieties of the detection probes used
in the methods of this invention are quenchers. The quencher can be
DABCYL, anthroquinone, nitrothiazole, nitroimidazole or malachite green.
Variants of DABCYL, such as DABSYL, DABMI or methyl red, are also
suitable. Also the asymmetric cyanine dye compounds disclosed in U.S.
Pat. No. 6,080,868 can be used as the quenching moiety; and are
incorporated by reference.

[0104] Additionally, fluorophores can also be used as the quenchers. For
example, fluorophores that do not fluoresce in the detection range when
the probe is in the open conformation can quench fluorescence when in
proximity with certain other fluorophores.

[0105] An example of a self-altering signal-generating probe is a
molecular beacon probe. The loop of a molecular beacon probe corresponds
to the probe sequence, as described above. Nucleotide sequences, referred
to as "arms," correspond to the first and second nucleotide sequences, as
described above. Molecular beacon probes are described in U.S. Pat. No.
5,925,517; PCT application WO95/13399; PCT application WO97/39008; and
Tyagi and Kramer (1996) Nature Biotechnology 14:303; and are incorporated
herein by reference.

[0106] Additionally, the molecular beacon probes can be modified in any
manner which allows for detection of the amplification products. Modified
probes include, for example, the "wavelength-shifting" molecular beacon
probes described in U.S. Pat. No. 6,037,130; and incorporated herein by
reference. In particular, these modified probes have the basic molecular
beacon probe structure, namely, a loop; stem duplex; a quencher on one
end; and a reporter moiety, typically a fluorophore, opposite the
quencher on the other end. The reporter is referred to as the "harvester
reporter." The modification of the probe is that the probe includes an
extension of several nucleotides past the "harvester reporter." The
extension terminates in a nucleotide that is linked to an "emitter
reporter," typically another fluorophore. In the presence of the target
nucleic acid molecule, the quencher separates from the reporters. In this
open conformation the "harvester reporter" absorbs energy from the
excitation source but transfers a significant portion of the energy, in
some constructions the great majority of the energy, to the "emitter
reporter," which receives the transferred energy and emits it at its
characteristic, longer wavelength.

[0107] In another embodiment, the detection probe includes a pair of
oligodeoxynucleotides complementary to contiguous regions of the
additional amplification products. (Cardullo et al. (1988), Proc. Nat'l.
Acad. Sci. 85: 8790-8794 and Heller et al. EP 00 70685. One
oligodeoxynucleotide contains the reporter moiety on its 5' end, and the
other oligodeoxynucleotide contains the interactive moiety on its 3' end.
When the probe is hybridized to the target sequence, the two moieties are
brought very close to each other. When the sample is stimulated by light
of an appropriate frequency, fluorescence resonance energy transfer from
one moiety to the other occurs, producing a measurable change in spectral
response from the moieties, thus signaling the presence of targets.

[0108] In yet another embodiment, the detection probe includes a pair of
oligodeoxynucleotides. The pair is complementary to one another. Also,
one of the pair has the sequence of the target nucleic acid molecule; and
the other of the pair has the sequence which is complementary to the
target nucleic acid molecule. (Morrison and Stols, "Sensitive
Fluorescence-Based Thermodynamic and Kinetic Measurements of DNA
Hybridization in Solution," Biochemistry 32: 309-3104 (1993) and Morrison
EP 0 232 967 A2, claiming priority of U.S. application Ser. No. 817,841,
filed Jan. 10, 1986.) Each oligodeoxynucleotide of the probe includes a
reporter moiety conjugated to its 3' end and an interactive moiety
conjugated to its 5' end. When the two oligonucleotides of the probe are
annealed to each other, the reporter moiety of each is held in close
proximity to the interactive moiety of the other. With the probe in this
conformation, if the reporter is then stimulated by light of an
appropriate wavelength, the signal is altered, preferably quenched, by
the interactive moiety. However, when either probe molecule is bound to a
target, the altering effect of the complementary oligodeoxynucleotide of
the probe is absent. In this conformation a signal is generated. The
oligodeoxynucleotides of the probe are too long to self-quench by FRET
when in the target-bound conformation.

[0109] The signal generated by the detection probe can be detected and
measured by any means known in the art which provides reliable detection
and measurement.

[0110] For example, the ABI 7700 (manufactured by Applied Biosystems, Inc.
in Foster City, Calif.) is adapted for measuring signal emission,
typically fluorescence emissions. The ABI 7700 uses fiber optics
connected with each well in a 96-well amplification reaction tube
arrangement. The instrument includes a laser for exciting the reporter
moieties and is capable of measuring the signal intensity, typically
fluorescence spectra intensity, from each tube with continuous monitoring
during amplification.

[0111] The additional amplification products can be quantified by endpoint
and real-time measurements. In an end-point mode, the signal measurement
is performed after the amplification reaction is complete, e.g., after
all or substantially all of the cycles of an amplification reaction have
been completed. In a real-time mode, signal measurement is performed
multiple times during the amplification reaction, e.g., after each
thermocycle of an amplification reaction. The real-time mode is preferred
when a quantitative measure of the initial amount of target nucleic acid
molecule is required, e.g., the copy-number of viral or bacterial nucleic
acids present in a sample.

[0112] The absolute amount of a target nucleic acid molecule present in a
test sample prior to amplification can be determined using a standard
curve. For example, a standard curve can be generated from the results
obtained from a series of parallel primer extension chain reactions.
These parallel reactions are performed on a series of standard samples
that contain a known amount of a nucleic acid molecule which is similar
to the target nucleic acid molecule. A series of about five to about
twenty standard samples of different known amounts are used. The parallel
extension reactions use the same reaction conditions and reagents as used
in the extension reaction of the target nucleic acid molecule.

[0113] In each parallel reaction, the increase in signal intensity as
compared with the baseline signal intensity (delta Rn) is measured at the
annealing temperature for each amplification cycle. The baseline value is
the magnitude of the signal detected prior to the formation of the
additional amplification products. Threshold values (Ct) are calculated
for each reaction. Ct is the amplification cycle number at which the
generated signal intensity is distinguishable from the baseline signal
intensity. The starting quantity of the nucleic acid in each standard
sample can be plotted against its corresponding Ct value. This plot is
the standard curve.

[0114] In general, the threshold value must be high enough to be
statistically different from the baseline value but below the signal
obtained for the saturation phenomenon associated with amplification
reactions. Typically, the threshold value is set at about ten standard
deviations above the mean baseline signal intensity. (See, for example,
Heid, et al. Genome Research 6:986-994 (1996)).

[0115] The Ct value for the sample including the target nucleic acid
molecule is also calculated. This Ct value can be plotted against the
standard curve. Using the standard curve, the amount of the target
nucleic acid molecule in the test sample can then be quantified by
extrapolation. FIG. 9 illustrates this method of PCR target quantitation
(in the case of HCV RNA) using the "nose-to-nose" PCR primers and
reaction conditions described in the Example below.

[0116] Computer software provided with detection instruments, for example
the ABI 7700, is capable of recording the signal intensity over the
course of an amplification. These recorded values can be used to
calculate the increase in signal intensity on a continuous basis.
Although the ABI 7700 instrument is typically used to monitor
fluorescence, the Ct values need not be determined from fluorescence
measurements. Ct values could be determined from measurements of a
variety of different types of signals.

[0117] The present invention also relates to kits, multicontainer units
comprising useful components for practicing the present method. The kit
comprises a set of nose-to-nose primers for the amplification of the
variants of a particular pathogen; and a probe, such as the self-altering
signal-emitting probes described above. In some cases, the probes are
fixed to an appropriate support membrane. Other optional components of
the kit include, for example, an agent to catalyze the synthesis of
primer extension products, the substrate nucleoside triphosphates, the
appropriate buffers for amplification and/or hybridization reactions, a
nucleic acid reference standard to permit quantitation of template
molecules in test samples, and instructions for carrying out the present
method.

EXAMPLES

[0118] The examples of the present invention presented below are provided
only for illustrative purposes and not to limit the scope of the
invention. Numerous embodiments of the invention within the scope of the
claims that follow the examples will be apparent to those of ordinary
skill in the art from reading the foregoing text and following examples.

Detection of HCV Variants

[0119] A comparison was made of three different methods for nucleic
acid-based detection of eight strains of HCV which are prototypes for the
main HCV genotypes and subtypes. The four detection methods used were:
(A.) The nose-to-nose beacon RT-PCR of the present invention, (B.)
Conventional beacon RT-PCR, and (C.) The COBAS AMPLICOR HCV MONITOR Test
Version 2.0 (COBAS HCM-2; Roche Diagnostic Systems Inc, Branchburg,
N.J.). The COBAS HCM-2, which is an RT-PCR-based assay, was carried out
according to the manufacturers' instructions. Conventional beacon RT-PCR
and "nose-to-nose" beacon RT-PCR were carried out as follows:

[0120] PCR primers were designed to amplify a segment of the 5' non-coding
region of the HCV genomic RNA. The nucleic acid sequence of this region
of the genome is relatively highly conserved between HCV genotypes and
subtypes.

[0121] Conventional primers for beacon PCR were designed to amplify a 101
b.p. segment of DNA corresponding to nucleotides 66 to 166 of the
published sequence of HCV-H [Inchauspe et al, Proc Natl Acad Sci (USA),
88:10292-10296, 1991; Genbank M67463]. The intervening gap between the
two conventional RT-PCR primers is 61 b.p. in length. The primers are as
follows:

[0122] The primers for "nose-to-nose" RT-PCR of the present invention were
designed such that there is no intervening nucleotide gap between the two
primers. The region amplified is a 51 b.p. segment corresponding to
nucleotides 83 to 133 of the published sequence of HCV-H. The primers are
as follows:

[0123] For both conventional and "nose-to-nose" PCR, the molecular beacon
used for PCR product detection was
5'-FAM-ccgggcTTAGTATGAGTGTCGTGCAGCCTgcccgg-DABCYL-3' (SEQ ID NO: 5). The
stem nucleic acids are shown in lower case, and the probe loop nucleic
acids (corresponding to nucleotides 91 to 113 of the HCV-H sequence) are
shown in upper case.

[0126] Table 1 shows a comparison of the three different methods for
detection of eight strains of HCV. These strains are prototypes for the
main HCV genotypes and subtypes. The nose-to-nose beacon assay of the
present invention (A) detects all eight genotypes/subtypes, while
conventional beacon PCR (B) fails to detect Genotypes 4a and 5a.

[0127] Results from the COBAS-HCM-2 assay are presented as International
Units (I.U.). Although various conversion factors have been suggested
(Saldanha et al, Vox Sang, 1999; 76(3):149-158; Cuijpers et al, 2001;
81(1):12-20), the exact relationship between I.U. and HCV RNA copy number
is still under debate, particularly for HCV genotypes other than 1a and
1b. Because of this, Roche Diagnostic Systems does not currently suggest
a conversion factor for results obtained with the COBAS-HCM-2 assay. For
analytical purposes, it is therefore assumed that I.U. and RNA copy
number are equivalent. In comparison with the COBAS-HCM-2 assay, the
nose-to-nose beacon RT-PCR assay is equivalent to or slightly more
sensitive for Genotypes 1a, 1b, 2b and 6a, but is 1 log (10-fold) more
sensitive for Genotype 4a, 0.5 log (3.2-fold) more sensitive for
Genotypes 2a and 5a, and 0.3 log (2-fold) more sensitive for Genotype 3a.
A statistical analysis comparing the relative sensitivity of the two
assays is shown in Table 2.

[0128] The "nose-to-nose" PCR of the present invention was used to screen
a subset of patient plasma samples from the ICBS HCV Master Panel. This
panel, which is being expanded continually, is compiled by the Centers
for Disease Control (CDC) in collaboration with the International
Consortium for Blood Safety (ICBS). The panel consists of plasma samples
collected from diverse geographic regions. All samples are screened for
HCV antibody and are subjected to genotype analysis in two independent
testing laboratories at the CDC and at Visible Genetics Inc. (VGI).

[0129] A total of 192 specimens, comprising plasma samples collected in
Egypt, Vietnam and Indonesia were provided by the CDC. Of these, 134 were
listed as being unequivocally positive for HCV RNA on the basis of
PCR-genotyping data obtained by CDC, VGI or both. Five samples were
listed as having equivocal or conflicting PCR-genotype data. Fifty-three
samples were listed as not genotypeable (i.e. negative for HCV RNA).

[0130] Total RNA was extracted from 70 μl freshly thawed plasma using a
robotic extraction procedure in which RNA is bound and eluted from PVDF
membranes in 96-well plate format (Lee and Prince, 2001, Transfusion;
41:483-487). Total RNA was obtained in a volume of 50 μl nuclease-free
water. Ten microliters of this (equivalent to 14 μl plasma) were then
subjected to reverse transcription and PCR using the nose-to-nose primers
(SEQ ID NO: 3 and SEQ ID NO: 4) and molecular beacon (SEQ ID NO: 5)
described above.

[0131] Table 3 shows the results of RT-PCR for the 134 unequivocally
HCV-positive samples present in the panel. "Nose-to-nose" PCR
successfully detected the vast majority of HCV isolates from all
genotypes. Of the 53 samples which were not positive for HCV RNA in
genotype assays, only one sample gave a weakly positive PCR signal
(103.1 RNA molecules per ml).

[0132] Samples with a virus burden less than ˜700 copies/ml (9.9
copies per 14 μl plasma) would not be detected using the combination
of robotic extraction and "nose-to-nose" RT-PCR describe above. The
results shown in Table 3 clearly demonstrate that the present invention
permits the detection of diverse HCV genotypes with a single set of
"nose-to-nose" primers and molecular beacon.

Comparison of Nose-to-Nose PCR and Conventional PCR for Detection of HIV-1
Group M Subtype B Variants

[0133] FIG. 6a shows an alignment of proviral DNA sequences corresponding
to the V3 region and flanking sequences of four different HIV variants,
all of Group M (Major) Subtype B (HIV/RT-1, HIV/RT10, HIV-38-1 and
HIV/38/3). The V3 region is the most highly variable segment of the HIV
genome. A molecular beacon was designed with a probe-loop structure
exactly identical to variant HIV/RT-1 (nucleotides 76-97 on the V3
sequence shown). This probe sequence possesses 1, 3, or 4 mismatches with
variants HIV/RT-10, HIV/38-1 and HIV/38-3 respectively (FIG. 6a).

[0134] PCR primers for "nose-to-nose" PCR were designed as follows. The
forward primer (5'-acaatacaagaaaaaggataactatgggac-3') (SEQ ID NO: 6)
corresponds to nucleotides 65-94 of the sequence of HIV/RT-1 shown in
FIG. 6a. The forward primer is known as NBF. The reverse primer (5'
tttctcctgttgtataaagtactctccccg-3') (SEQ ID NO: 7) corresponds to
nucleotides 95-124 of the same sequence. The reverse primer is known as
NBR.

[0135] Primers for conventional PCR were designed to generate a 177 b.p.
PCR product as follows. The forward primer
(5'taatagtacagctgaatgaatctg-3') (SEQ ID NO: 8) corresponds to nucleotides
14-37 of the sequence of HIV/RT-1 shown in FIG. 6a. The reverse primer
(5'gttttaaagtgttattccatgc-3') (SEQ ID NO: 9) corresponds to nucleotides
168-190 of the same sequence.

[0136] FIG. 7 shows the results of an experiment to compare the ability of
conventional beacon PCR with nose-to-nose PCR for detection of each of
the 4 HIV variants shown in FIG. 6a. PCR reactions contained 106
template molecules of HIV/RT-1, HIV/RT-10, HIV/38-1 or HIV/38-3, the
molecular beacon shown in FIG. 6b, and either the conventional or
nose-to-nose primers described above. Control PCR reaction contained
either no template or 150 ng human genomic DNA. Amplification was
performed in the Perkin Elmer 7700 using the following cycling
parameters: 95° C. for 10 min, followed by 40 cycles of 95°
C. for 30 sec (denaturation), 50° C. for 1 min (annealing) and
72° C. for 30 sec (extension). Fluorescence of the molecular
beacon was measured at the 50° C. annealing temperature.
Fluorescence was then plotted graphically against PCR cycle number.
Efficiency of PCR amplification/detection is determined by the "threshold
cycle" i.e. the lowest numbered PCR cycle required to generate a positive
fluorescent signal.

[0137] As shown in FIG. 7a, the conventional beacon PCR technique was
capable of detecting both HIV/RT-1 (exact match to the beacon) and
HIV/RT-10 (one mismatch), with an equivalent threshold cycle (cycle 23)
although the peak level of fluorescence obtained in the latter case was
˜2 fold lower than that for the exact-match template. The
conventional beacon PCR technique failed to detect either HIV/38-1 (3
mismatches) or HIV/38-3 (4 mismatches), despite the fact that PCR product
was generated from all 4 variants, as shown by gel analysis (FIG. 8).

[0139] PCR primers were designed to amplify a segment of the gag gene of
the HIV-1 genomic RNA, which is relatively well conserved between the
different Subtypes of HIV-1 Group M. Despite this relative conservation,
individual HIV-1 Subtypes show up 20% nucleotide sequence diversity
within this region of the genome (Roberton et al, 1999 in: Human
Retroviruses and AIDS 1999, pp 492-505, Editors Kuiken et al, Los Alamos
National Laboratory, Los Alamos, N. Mex.).

[0140] Conventional primers for beacon PCR were designed to amplify a 94
b.p. segment of RNA corresponding to nucleotides 1478 to 1571 of the
published sequence of the Subtype B HIV-1 isolate HXB2 [Ratner et al,
1985, Nature, 313(6000):277-284; Genbank K03455]. The intervening gap
between the two conventional RT-PCR primers is 53 b.p. The primers are as
follows:

[0141] The primers for "nose-to-nose" RT-PCR of the present invention were
designed to amply a 57 b.p. segment corresponding to nucleotides 1502 to
1558 of the published sequence of HIV-1 HXB2. There is no intervening gap
between the two primers. The primers are as follows

[0142] For both conventional and "nose-to-nose" PCR, the molecular beacon
used for PCR product detection was
5'-FAM-cgcctTACCCTTCAGGAACAAATAGaggcg-DABCYL-3' (SEQ ID NO: 14). The stem
nucleic acids are shown in lower case, and the probe loop nucleic acids
(corresponding to nucleotides 1512 to 1530 of the HIV-1 HXB2 sequence)
are shown in upper case.

[0143] Virus isolates from HIV-1 Subtypes A, B, C, D, F and G were
obtained as cell-free culture supernatants from the AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID, NIH. RNA was
extracted from 140 μl freshly thawed culture supernatant.
Complementary DNA was reverse transcribed from extracted RNA using either
the conventional reverse primer, or the nose-to-nose reverse primer
described above. Reaction conditions for both cDNA synthesis and PCR
amplification were essentially the same as those described above for HCV.
All assays were carried out in triplicate. Quantitation of HIV template
molecules was achieved by inclusion of an RNA standard curve in each
RT-PCR experiment. The standard curve was constructed using 1, 10,
102, 103, 104, 105 or 106 molecules HIV-1 RNA
diluted in 1 mg/ml yeast tRNA (Ambion, Austin, Tex.).

[0144] Table 4 shows a comparison of conventional RT-PCR and
"nose-to-nose" RT-PCR for detection of HIV-1 Subtypes. The "nose-to-nose"
assay of the present invention (A.) detects all 6 HIV-1 Subtypes tested,
while the conventional assay fails to detect Subtypes A, D and G.

Adaptation of the Method for Simultaneous Detection of Both HIV-1 Group M
and HIV-1 Group O Variants

[0145] Virus isolates from HIV-1 Group 0 (Outlier) show marked sequence
variation from members of HIV-1 Group M (Major). Although Group 0 viruses
are mainly prevalent in parts of Africa, their frequency among samples
collected by blood banks outside Africa appears to be increasing (Jaffe
and Schochetman, 1998, Infect Dis Clin North Am; 12(1):39-46; Couturier
et al, 2000, AIDS; 14(3):289-296; Fed Regist, 1997, Sep. 23;
62(184):49695). The present invention permits detection of members of
both Group M and Group 0 using a single set of "nose-to-nose" primers and
molecular beacon.

[0146] The primers for "nose-to-nose" RT-PCR of the present invention were
designed to amplify a 64 b.p. segment of the pol gene of the HIV-1
genomic RNA, corresponding to nucleotides 4750 to 4813 of the published
sequence of HIV-1 HXB2. There is no intervening gap between the two
primers. The primers are as follows

[0147] The molecular beacon used for PCR product detection was
5'-FAM-cgcacgGCAGTATTCATTCACCAATTTTcgtgcg-DABCYL-3' (SEQ ID NO: 17). The
stem nucleic acids are shown in lower case, and the probe loop nucleic
acids are shown in upper case.

[0148] The new primers and beacon were then tested for their ability to
amplify and detect virus isolates from HIV-1 Group M (Subtypes A, B, C,
D, F and G) and Group O, which were obtained as cell-free culture
supernatants from the AIDS Research and Reference Reagent Program,
Division of AIDS, NIAID/NIH. Prior to RNA extraction, all cell
supernatants were diluted 1000-fold in phosphate buffered saline (PBS).
RNA extraction was performed on the diluted supernatant essentially as
described above, except that following isolation, all RNA samples were
treated with RNase-free DNase (Ambion, Austin, Tex.) to ensure removal of
contaminating proviral DNA. Complementary DNA was reverse transcribed
from extracted RNA using the nose-to-nose reverse primer (SEQ ID NO: 16)
described above. Reaction conditions for both cDNA synthesis and PCR
amplification were essentially the same as those described above.
Quantitation of HIV-1 template molecules was achieved by inclusion of an
RNA standard curve in each RT-PCR experiment.

[0149] Table 5 shows the results of RT-PCR using the pol region
"nose-to-nose" primers of the present invention, and a comparison with
the COBAS AMPLICOR HIV-1 Monitor Assay Version 1.0 (Roche Diagnostics,
Branchburg, N.J.). The "nose-to-nose" RT-PCR assay was capable of
detecting all Group M and Group 0 isolates tested, with sensitivity equal
to (Group M, Subtypes B and C) or greater than (Group M, Subtypes A, D
and F) the COBAS AMPLICOR HIV-1 Monitor Assay (1.0). The latter assay
failed to detect either of the Group 0 virus isolates tested, and also
failed to detect Group M Subtype G. These data are in agreement with a
recent report that neither the COBAS AMPLICOR HIV-1 Monitor Assay (1.0)
nor its improved version (1.5) are capable of detecting group 0 viruses
(Yang et al, Transfusion, 2001; 41:643-651). In contrast, the present
invention permits detection of all virus isolates with a single set of
"nose-to-nose" primers and molecular beacon.

[0150] Unless otherwise indicated, all numbers expressing quantities of
ingredients, properties such as molecular weight, reaction conditions,
and so forth used in the specification and claims are to be understood as
being modified in all instances by the term "about." Accordingly, unless
indicated to the contrary, the numerical parameters set forth in the
specification and attached claims are approximations that may vary
depending upon the desired properties sought to be obtained by the
present invention. At the very least, and not as an attempt to limit the
application of the doctrine of equivalents to the scope of the claims,
each numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary rounding
techniques. Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations, the
numerical values set forth in the specific examples are reported as
precisely as possible. Any numerical value, however, inherently contains
certain errors necessarily resulting from the standard deviation found in
their respective testing measurements.

[0151] The terms "a," "an," "the" and similar referents used in the
context of describing the invention (especially in the context of the
following claims) are to be construed to cover both the singular and the
plural, unless otherwise indicated herein or clearly contradicted by
context. Recitation of ranges of values herein is merely intended to
serve as a shorthand method of referring individually to each separate
value falling within the range. Unless otherwise indicated herein, each
individual value is incorporated into the specification as if it were
individually recited herein. All methods described herein can be
performed in any suitable order unless otherwise indicated herein or
otherwise clearly contradicted by context. The use of any and all
examples, or exemplary language (e.g., "such as") provided herein is
intended merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed. No language
in the specification should be construed as indicating any non-claimed
element essential to the practice of the invention.

[0152] Groupings of alternative elements or embodiments of the invention
disclosed herein are not to be construed as limitations. Each group
member may be referred to and claimed individually or in any combination
with other members of the group or other elements found herein. It is
anticipated that one or more members of a group may be included in, or
deleted from, a group for reasons of convenience and/or patentability.
When any such inclusion or deletion occurs, the specification is deemed
to contain the group as modified thus fulfilling the written description
of all Markush groups used in the appended claims.

[0153] Certain embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Of course, variations on these described embodiments will
become apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventor expects skilled artisans to employ
such variations as appropriate, and the inventors intend for the
invention to be practiced otherwise than specifically described herein.
Accordingly, this invention includes all modifications and equivalents of
the subject matter recited in the claims appended hereto as permitted by
applicable law. Moreover, any combination of the above-described elements
in all possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by context.

[0154] Specific embodiments disclosed herein may be further limited in the
claims using consisting of or consisting essentially of language. When
used in the claims, whether as filed or added per amendment, the
transition term "consisting of" excludes any element, step, or ingredient
not specified in the claims. The transition term "consisting essentially
of" limits the scope of a claim to the specified materials or steps and
those that do not materially affect the basic and novel
characteristic(s). Embodiments of the invention so claimed are inherently
or expressly described and enabled herein.

[0155] Furthermore, numerous references have been made to patents and
printed publications throughout this specification. Each of the
above-cited references and printed publications are individually
incorporated herein by reference in their entirety.

[0156] In closing, it is to be understood that the embodiments of the
invention disclosed herein are illustrative of the principles of the
present invention. Other modifications that may be employed are within
the scope of the invention. Thus, by way of example, but not of
limitation, alternative configurations of the present invention may be
utilized in accordance with the teachings herein. Accordingly, the
present invention is not limited to that precisely as shown and
described.